EPIGALLOCATECHIN GALLATE AS A NEUROPROTECTANT IN A MODEL OF MULTIPLE SCLEROSIS

描述（由申请人提供）：（-）-表没食子儿茶素-3-没食子酸酯（EGCG）是多酚化合物的黄烷-3-醇家族的有效抗氧化剂，并且发现富集在来自植物茶树的绿色茶中。表没食子儿茶素没食子酸酯是实验性自身免疫性脑脊髓炎（EAE）的有效抑制剂，EAE是多发性硬化症的模型。本提案的具体目的是寻求提供设计EGCG治疗多发性硬化症的临床试验所需的临床前数据，并探索其神经保护作用机制。该提案有两个具体目标：1）验证EGCG抑制EAE，并在存在炎症浸润的情况下具有神经保护作用，并确定口服给予EGCG的药代动力学; 2）确定EGCG是否通过抑制线粒体介导的细胞死亡途径来保护SH-SY 5 Y神经母细胞瘤细胞。将在饮用水中施用不同浓度的EGCG后测定EGCG对EAE的影响。将通过甲苯胺蓝染色后的形态测定分析对来自处理和未处理小鼠的脊髓的白色物质中的脱髓鞘和轴突损伤进行病理学评估，并通过磷酸化神经丝的免疫组织化学检测确认轴突损伤。将通过免疫组织化学分析进行炎性细胞浸润的表型和定量。接受不同剂量的EGCG的小鼠中的EGCG血浆水平将通过高效液相色谱法测定，并且血浆水平将与临床和组织学益处相关。EGCG的神经保护作用的体外检查将包括用以下物质诱导细胞死亡：TNF-α、L-谷氨酸、NO供体（NOC-12）和过氧化氢。将通过测量胞质细胞色素c、切割和未切割的半胱天冬酶-9、-3和-8以及切割和未切割的Bid来评估外源性和内源性细胞死亡途径，以确定EGCG在什么点抑制细胞死亡。与公共卫生的相关性：我们正在评估EGCG的神经保护能力，通过评估其在MS动物模型中的作用并表征其体外作用机制。本研究的目的是为开发EGCG作为人类疾病多发性硬化症的新型神经保护治疗提供必要的基础。

DESCRIPTION (provided by applicant): (-)-Epigallocatechin-3-gallate (EGCG) is a potent antioxidant of the flavan-3-ol family of polyphenolic compounds and is found enriched in green tea from the plant Camellia sinensis. EGCG is a potent inhibitor of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The specific aims of this proposal seek to provide the pre-clinical data needed to design clinical trials of EGCG in multiple sclerosis and to explore its neuroprotective mechanism of action. The proposal has two specific aims: 1) To verify that EGCG inhibits EAE and is neuroprotective in the presence of inflammatory infiltrate, and to determine the pharmacokinetics of orally administered EGCG; 2) To determine whether EGCG protects SH-SY5Y neuroblastoma cells by inhibition of the mitochondrial mediated cell death pathways. The effects of EGCG on EAE will be determined following administration of varying concentrations of EGCG in drinking water. Pathological assessments will be made of demyelination and axonal damage in white matter of spinal cords from treated and untreated mice by morphometric analysis following toluidine blue staining and confirmation of axonal injury by immunohistochemical detection of phosphorylated neurofilaments. Phenotype and quantification of inflammatory cell infiltrate will be performed by immunohistochemical analyses. Plasma levels of EGCG in mice receiving varying doses of EGCG will be determined by high performance liquid chromatography and the plasma levels will be correlated with clinical and histologic benefit. In vitro examination of the neuroprotective effects of EGCG will include induction of cell death with the following: TNF-alpha, L-glutamic acid, an NO donor (NOC-12), and hydrogen peroxide. Extrinsic and intrinsic cell death pathways will be assessed by the measuring cytosolic cytochrome c, cleaved and uncleaved caspases-9, -3, and -8, and cleaved and uncleaved Bid to determine at what point EGCG inhibits cell death. Relevance to public health: We are assessing the neuroprotective capacity of EGCG by assessing its effects in an animal model of MS and characterizing its mechanism of action in vitro. The purpose of this research is to provide the groundwork necessary to develop EGCG as a novel neuroprotective treatment for the human disease, multiple sclerosis.